This proposal has two primary objectives: 1) The large scale production of erythropoietin by kidney cells in culture and 2) The study of mechanisms regulating formation and secretion of the hormone. Some existing evidence indicates that small but significant amounts of biologically active erythropoietin can be formed by bovine, rat and human embryonic kidney cells in culture. We propose an intensive study of the conditions required for maximal production. Cells will be exposed to lowered oxygen tension, cobaltous salts, and anabolic steroids as well as to cyclic AMP, and prostaglandins. All of these have been shown to exert an effect on erythropoietin formation in vivo. In addition, we plan to use standard cloning techniques and to develop methods of fractionation of kidney cells in order to purify the population responsible for erythropoietin formation, as a preliminary step in establishing a line of cells capable of sustained hormone production and release.